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Dimer formation of a stabilized Gbeta1 variant: a structural and energetic analysis
Stephanie Thoms1, Klaas E A Max, Michael Wunderlich
1Universität Bayreuth, Germany.
Journal of Molecular Biology
|June 17, 2009
Summary
Mutations in streptococcal protein G beta1 domain (Gbeta1) enhance stability. Specific substitutions enlarge the hydrophobic core, leading to dimer formation through intermolecular hydrogen bonds and hydrophobic interactions.
Area of Science:
- Protein engineering
- Biophysics
- Structural biology
Background:
- Streptococcal protein G beta1 domain (Gbeta1) is a model system for protein stability studies.
- Previous work generated a stabilized Gbeta1 variant (Gbeta1-M2) using in vitro selection.
- Gbeta1-M2 contains four specific amino acid substitutions: E15V, T16L, T18I, and N37L.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying the enhanced stability of Gbeta1-M2.
- To analyze the individual and combined contributions of the four Gbeta1-M2 substitutions to protein stabilization.
- To understand the structural basis for dimer formation in the stabilized Gbeta1 variant.
Main Methods:
- Site-directed mutagenesis and in vitro selection were used to generate and analyze Gbeta1 variants.
- Thermodynamic stability was assessed by measuring unfolding.
- Analytical ultracentrifugation and NMR spectroscopy were employed to study protein-protein interactions.
- X-ray crystallography was used to determine the high-resolution structure of Gbeta1-M2.
Main Results:
- All four single substitutions (E15V, T16L, T18I, N37L) individually stabilize wild-type Gbeta1.
- Substitutions T16L and N37L significantly contribute to stabilization by expanding the hydrophobic core.
- These substitutions promote dimer formation, evidenced by concentration-dependent stability and analytical ultracentrifugation.
- The crystal structure reveals head-to-head dimer formation mediated by intermolecular hydrogen bonds and a hydrophobic interface.
Conclusions:
- The enhanced stability of Gbeta1-M2 results from specific amino acid substitutions that enlarge the hydrophobic core.
- These substitutions induce dimer formation through novel intermolecular interactions.
- The findings provide insights into protein stabilization strategies and the structural basis of protein-protein interactions.
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